Bystander response to 0.5 Gy of alpha-particles in a human 3-dimensional skin model in 16h after exposure to ionizing radiation. Homo sapiens

Ionizing radiation (IR) not only affects cells that are directly irradiated but also their non-irradiated neighbors, which show responses known as bystander effects. While bystander and direct responses have several common end points including apoptosis and micronucleation, chromatin remodeling and altered levels or activities of regulatory proteins, they can be quantitatively and qualitatively different. The majority of studies of radiation bystander effects have utilized 2-dimensional in vitro systems, but we have recently demonstrated such effects in EPI-200 (Mat-Tek, Ashland, MA), a 3-dimensional tissue model that precisely imitates the structure and function of human epidermis. Global gene expression is a powerful tool for uncovering both fundamental signaling processes and the mechanistic basis of cellular or physiological effects. By exposing only a thin strip across the center of the EPI-200 tissue, we have been able to measure global gene expression responses in bystander cells located at 0.125 and 0.625 um from the irradiation line, in 16h after irradiation. The data were analyzed using BRB-Array Tools (NIH), and further network analysis was performed with IPA (Ingenuity). Significantly responding genes were identified at the both distances. For instance, all sets demonstrated upregulation of two key enzymes of the lipid biosynthesis, UGT1 and PITPNB, and downregulation of proapoptotic proteins: BAX and ARHGEF5. In contrast, several proteins involved in transcriptional repression (CHD6, CHD8 andWRNIP1) were strongly upregulated suggesting a rearrangement in the gene transcription. These changes suggest an activation of bystander mechanisms different from those observed in 2-dimensional cell cultures. Overall design: Radiation induced gene expression in 3-dimensional tissue model, Epi-200, was measured in 16 hours after exposure to 0.5 Gy of alpha-particles. Three independent experiments were performed for the samples collected at different distances from the irradiation line (125-625 and 625-1125 um) using three tissue fragments per a data point.

电离辐射（Ionizing radiation, IR）不仅会直接作用于受照射的细胞，还会对其未受照射的邻近细胞产生影响，这类邻近细胞所产生的应答被称为旁效应（bystander effects）。尽管旁效应与直接照射应答存在多项共同终点，包括细胞凋亡、微核形成、染色质重塑以及调控蛋白的水平或活性改变，但二者在定量与定性层面均存在显著差异。当前绝大多数辐射旁效应相关研究均采用二维体外培养系统，而本团队近期在EPI-200（Mat-Tek，美国马萨诸塞州阿什兰市）——一种可精准模拟人类表皮结构与功能的三维组织模型——中证实了这类效应的存在。全局基因表达分析是揭示基础信号转导过程以及细胞或生理效应分子机制的有力工具。本研究仅对EPI-200组织中央的窄条区域进行照射，得以检测照射后16小时内，距照射线0.125与0.625 μm处的旁效应细胞的全局基因表达应答。数据采用BRB-Array Tools（美国国立卫生研究院，NIH）进行分析，并通过IPA（Ingenuity系统）开展后续网络分析。在两个距离处均鉴定到了显著响应的基因。例如，所有样本组均显示脂质生物合成的两种关键酶UGT1与PITPNB表达上调，而促凋亡蛋白BAX与ARHGEF5表达下调。与之相反，多种参与转录抑制的蛋白（CHD6、CHD8及WRNIP1）出现显著上调，这提示基因转录过程发生了重排。上述变化表明，三维组织模型中的旁效应激活机制与二维细胞培养体系中观察到的机制存在显著差异。整体实验设计：本研究检测了三维组织模型EPI-200在暴露于0.5 Gy α粒子后16小时内的辐射诱导基因表达情况。针对距照射线不同距离（125~625 μm与625~1125 μm）收集的样本，开展了三次独立实验，每个数据点对应三份组织片段。